Summary Our long-term goal is to develop a novel treatment for Tuberculosis (TB) which is one of the most devastating diseases worldwide, infecting ~1/3 of the global population and claiming more than ~1.5 million lives each year. The shortcomings of currently available TB drugs underscore the urgent need to discover novel compounds to effectively treat TB patients. The current ?short course? front-line regimen involves a cocktail of multiple drugs taken for 6-9 months. This protracted treatment stems from the difficulty of eradicating dormant populations of Mtb in various niches throughout the body. The emergence of Multidrug-Resistant (MDR-TB) and Extremely Drug Resistant (XDR-TB) strains of TB further complicates the control of this disease. Thus, there is an urgent need for potent drugs with novel modes of action capable of shortening the course of treatment and killing drug-resistant and dormant Mtb in vivo. To address this problem we will combine the chemical diversity of natural products (NP) and our capability to conduct whole-cell drug screening against Mtb under in vivo-like conditions to identify novel scaffolds to fuel the TB drug development pipeline. Encouraged by our discovery of numerous fungal NP active against both replicating and dormant Mtb in our screens of ~2500 fungal extracts, in Aim 1 we propose to characterize the potency and selectivity of active NP to identify high priority samples for deconvolution and identification of novel scaffolds with anti-TB activity. Our preliminary data support the hypothesis that screening NP samples under in vivo- like conditions such as our in vitro dormancy model can reveal ?hit? compounds presumably acting on novel targets that are only essential, and thus vulnerable to inhibition, under stress conditions encountered in vivo. Aim 2 will focus on purifying the active components from the most potent and selective mixtures, determining their chemical structure, and conducting comprehensive analysis of antimicrobial activity and physicochemical properties. Potent and selective inhibitors of dormant, intracellular, and/or drug resistant Mtb that we identify in this project will serve as the basis for future hit-to-lead development of novel candidate therapeutics for TB.